A Single-Molecule Strategy to Capture Non-native Intramolecular and Intermolecular Protein Disulfide Bridges

Non-native disulfide bonds are dynamic covalent bridges that form post-translationally between two cysteines within the same protein (intramolecular) or with a neighboring protein (intermolecular), frequently due to changes in the cellular redox potential. The reversible formation of non-native disulfides is intimately linked to alterations in protein function; while they can provide a mechanism to protect against cysteine overoxidation, they are also involved in the early stages of protein multimerization, a hallmark of several protein aggregation diseases. Yet their identification using current protein chemistry technology remains challenging, mainly because of their fleeting reactivity. Here, we use single-molecule spectroscopy AFM and molecular dynamics simulations to capture both intra- and intermolecular disulfide bonds in γD-crystallin, a cysteine-rich, structural human lens protein involved in age-related eye cataracts. Our approach showcases the power of mechanical force as a conformational probe in dynamically evolving proteins and presents a platform to detect non-native disulfide bridges with single-molecule resolution.


■ INTRODUCTION
Native disulfide bridgesstrong covalent bonds formed between two close cysteine residueshave been typically shown to provide structural stabilization to the folded conformation of proteins. 1,2 In addition to those disulfides with a clear structuralstaticfunction, other intramolecular disulfide bonds display a dynamic behavior in virtue of their reversible thiol/disulfide chemistry, a mechanism that is collectively related to a myriad of redox-mediated signaling cellular processes, 3−5 and tightly modulated by the fluctuating redox properties of the environment and by dedicated oxidoreductase enzymes that ensure overall redox homeostasis. 6 A further layer of complexity is added when solventexposed cysteines establish intermolecular disulfide bonds with a neighboring protein. 7−9 While intermolecular disulfide bonds play decisive functional rolesfor example, they work as protective mechanisms against cysteine irreversible overoxidation 10,11 but are also involved in a large number of aggregation misfolding reactions 12−14 their extremely fleeting nature makes them challenging to detect experimentally. Several techniques, such as NMR have attempted to capture their presence in solution, 15 and a handful of crystal structures have provided the snapshots of a subset of (long-lived) stable conformations. 16 However, their markedly fast reactivity, underpinning their ability to multimerize in a (often) rather uncontrolled way, calls for the development of new experimental approaches enabling direct detection of nonnative disulfide bonds.
Single-molecule force spectroscopy AFM has emerged as a powerful technique to identify intramolecular disulfide bonds, 17 which work as strong intramolecular staples. Typically, stretching a protein with an AFM extends it to almost its full contour length. 18 The presence of an intramolecular disulfide bond effectively shortcuts the protein, limiting its extensibility. 19 Hence, a shorter than expected increment of the protein's contour length can be considered as a reliable reporter of the presence of intramolecular disulfide bridges, as demonstrated in titin, 20,21 calmodulin binding domain, 22 FimG domain 23 and cell adhesion molecule domains. 24 Recent nanomechanical experiments also captured the dynamics of reduction and reformation of individual intramolecular disulfides by measuring the changes in protein extensibility over time. 25−30 Despite the unquestionable progress, this single-molecule approach is still not suitable to capture intermolecular, non-native disulfide bonds, notably because of the natural requirement of establishing a physical connection between (at least) two contacting proteins in the assay. Here, using a combination of protein engineering techniques (based on the rational design of polyproteins whereby the number of adjacent monomers prone to oxidation is precisely controlled), molecular dynamics (MD) simulations, and single-molecule force spectroscopy experiments, we develop an integrated single-molecule experimental approach able to capture both non-native inter-and intramolecular individual disulfide bonds, and characterize their dynamics under biologically mimicking redox conditions. We showcase our proof-of-principle experimental platform to study the complex redox reactivity of human γD-crystallin (γDc, a key human structural protein in the eye lens containing 6 reduced native cysteines), the aggregation of which 31−33 intricately related to cysteine oxidation 32 gives rise to the highmolecular weight protein aggregates that hallmark the eye's cataract disease. 34,35 Noteworthy, the ability to identify and trap non-native intermolecular disulfide bonds fingerprints a conformational change, involving the spontaneous and relatively frequent excursion of the native γD-crystallin to a well-defined intermediate conformation, that exposes to the solvent those natively cryptic (and thus unreactive) cysteines, becoming suddenly reactive. Besides enabling direct identification of non-native (intramolecular and intermolecular) disulfide bonds, our single-molecule strategy unambiguously captures disulfide-bond mediated protein dimerization and provides a direct probe of the subtle interplay between the redox status of a protein and its conformational dynamics.

■ RESULTS
Human γD-crystallin (γDc) is the third most abundant crystallin in the human lens, 36 which folds in the characteristic crystallin two-domain structure (Figure 1a). Despite its high degree of structural symmetry provided by its characteristic two Greek-key motifs, its cysteine content is not evenly distributed among the terminals. While the N-terminal domain (Ntd) harbors 4 cysteines, the C-terminal domain (Ctd) contains only two cysteines, arranged in a CXC motif, Figure  1b. In neither termini does the crystallized native structure show the presence of a disulfide bridge (PDB: 1HK0). Consequently, mechanical unfolding and stretching of a single γDc monomer with an AFM should uncomplicatedly elicit the whole length of the protein. To test this hypothesis, we first engineered a polyprotein construct whereby two γDc monomers are intercalated with the Ig91 marker protein, (Ig91−γDc) 2 ( Figure 2a). This strategy enables the marker protein not only to serve as an internal mechanical fingerprint (the mechanical properties of the Ig91 protein have been wellcharacterized, 18 Supporting Figures 1 and 2) but also to avoid any physical interaction between the two γDc monomers. Stretching an individual (Ig91−γDc) 2 polyprotein at a constant velocity of 400 nm s −1 with an AFM commonly resulted in unfolding trajectories with (up to) four unfolding peaks prior to the two unfolding peaks of the Ig91 markers (Figure 2b, top). Each of the (Ntd and Ctd) termini within each γDc monomer unfolded independently, and the mechanical unfolding of the full (Ig91−γDc) 2 construct quantitatively agreed in terms of unfolding forces (F Ntd = 131 ± 15 pN; F Ctd = 100 ± 19 pN) and contour length increments (ΔL c,Ntd = 29.0 ± 1.1 nm; ΔL c,Ctd = 29.7 ± 1.0 nm) with the independent mechanical characterization of each individual terminus (Supporting Figure 3). Consequently, in the full (Ig91−γDc) 2 typical unfolding trajectory, the first two peaks (Figure 2b, top) corresponded to the unfolding of the mechanically weaker Ctds, followed by the unfolding of the two Ntds, displaying higher mechanical stability, before the unfolding of the higher mechanical stability Ig91 markers.
As expected, in the vast majority of the unfolding traces (91%) (Figure 2b, top) the total full γDc length ΔL c ∼120 nm is released upon unfolding, since each γDc terminal contributes with ∼30 nm (4 termini × 30 nm = 120 nm, Supporting Figure  4). However, a few yet significant (9%) unfolding trajectories showed a marked reduction in the total (γDc) 2 ΔL c unfolding length, (γDc) 2 ΔL c < 120 nm (Figure 2b  Greek key motifs of the γDc (motifs I−IV), consisting of two intercalated antiparallel β-sheet motifs in each termini. Each red circle marks the location of the cysteines within the protein structure. native crystal structure ( Figure 1). Close inspection to the contour length histogram associated with crystallin unfolding ( Figure 2d) revealed a predominant peak at ∼30 nm, likely corresponding to the all-or-none unfolding of each individual domain, in addition to two extra lower occurrence peaks at shorter lengths (ΔL c ∼15 and ∼9 nm) that correspond to the unfolding of well-defined partially folded conformations occurring in the Ntd (ΔL c ∼15 nm and ∼130 pN) or the Ctd (ΔL c ∼9 nm and ∼155 pN), clearly distinguishable when pulling both γDc terminals independently (Supporting Figure  3). Remarkably, the relative abundance of both peaks is dramatically reduced under reducing conditions, while the probability of observing a ∼30 nm full-length unfolding event is instead increased. Combined, these experiments suggest that those shorter trajectories (Figure 2b, bottom) leading to protein extensions shorter than ∼120 nm ( Figure 2e) are directly related to the mechanical intermediate conformations that largely resolve upon GSH addition.
Having observed that γDc forms non-native intramolecular disulfide bonds, we questioned whether γDc can also establish intermolecular disulfide bonds with neighboring monomers. To this purpose, we built a polyprotein construct whereby two γDc monomers are physically connected to each other and flanked by the Ig91 marker proteins (Figure 3a). Pulling on the resulting Ig91−(γDc) 2 −Ig91 polyprotein resulted in a rich repertoire of unfolding trajectories, that could be classified into three mechanical phenotypes. In most cases (55%, Figure 3b, top), a regular (and expected) full-length unfolding trajectory consisting of four independent unfolding events of ∼30 nm (Supporting Figure 7) was observed. We also identified two distinct unexpected unfolding pathways that markedly departed from this regular behavior. First, around 23% of the trajectories exhibited a well-defined unfolding pattern ( Figure  3b, middle), where the typical mechanical hierarchy is lost, exhibiting unfolding peaks of alternating higher and lower mechanical stabilities. This unfolding scenario is overall compatible with γDc dimerization through a domain swap mechanism via Ntd interchange that we described earlier. 37 Importantly, the unfolding pattern does not exhibit any shortening in the (γDc) 2 length, implying that domain swapmediated dimerization maintains the native redox status of all 12 cysteines comprised in the two γDc monomers (Supporting Figure 8). The remaining 22% of the unfolding trajectories featured a significant decrease in the total (γDc) 2 unfolding length (Figure 3b, bottom), which is likely to be underpinned by the formation of non-native disulfide bridges. Direct comparison of the measured total ΔL c (γDc) 2 for the Ig91− (γDc) 2 −Ig91 polyprotein and the previously characterized (Ig91−γDc) 2 construct enabled us to directly identify whether the formation of non-native disulfide bridges occurs within the same protein or between the two adjacent γDc monomers, Figure 3c. We conclude that, when the total ΔL c (γDc) 2 is Examining the nanomechanical response of the same Ig91− (γDc) 2 −Ig91 polyprotein in the presence of reduced GSH (and TCEP, Supporting Figure 9) resulted in the absence of the shorter unfolding length phenotype, confirming that the formation of non-native disulfide bonds account for the The percentage values in brackets represent the frequency of each length phenotype. The regular unfolding trajectories (top) exhibit the initial unfolding of the two γDc monomers, releasing a total length of 120 nm. The non-oxidative dimerization length phenotype (middle) usually entails four γDc unfolding events with varying ΔL c values and mechanical stabilities. The overall total ΔL c (γDc) 2 extension is larger than the regular phenotype by 7 nm. (c) Histogram comparing the total γDc (ΔL c ) 2 when pulling Ig91− (γDc WT ) 2 −Ig91 (turquoise) and (Ig91−γDc WT ) 2 (gray) enabling to discriminate between (red vertical line) intramolecular (total γDc (ΔL c ) 2 > 80 nm) and intermolecular (total γDc (ΔL c ) 2 < 80 nm) non-native disulfide bridge formation in the absence of GSH. The non-native disulfide length phenotype (middle) typically displays a shorter total ΔL c (γDc) 2  shortening in (γDc) 2 extension. Under these reducing conditions, the two other unfolding phenotypes, namely the regular (Figure 3d, top) and the non-oxidative dimerization pathway (Figure 3d, bottom) were observed in an almost equal probability ( Figure 3e). As before, the histogram of γDc ΔL c clearly shows that the population of partially folded conformations vanishes after exposure to reducing conditions, while the ΔL c defining the domain swapped conformation increases its probability of occurrence (Figure 3f).
The first obvious and unavoidable requirement for the formation of intermolecular disulfide bonds is that the involved cysteines from both neighboring proteins are surface-exposed. To evaluate the degree of sulfur exposure of the 6 different cysteines in each γDc monomer we conducted MD simulations, which demonstrated that only the sulfur in cys 111 (in the Ctd) is partially exposed to the environment, while the other 5 cysteines are completely buried in the folded structure (Figure 3g). This finding suggests that, for the intermolecular disulfide bonds to occur, the individual γDc monomers need to undergo a conformational change (in the absence of force) that destabilizes the protein, ultimately resulting in cysteine exposure to the solvent. With this rationale in mind, we aimed to elucidate whether protein destabilization enhances non-native intermolecular disulfide formation. Given that 4 of the cysteines in γDc are present in the N-terminal, where, incidentally, most of the congenital cataract-point mutations cluster, 38,39 we hypothesized that the introduction of single-point mutations in the Ntd could lead to destabilization, hence increasing the probability of cysteine exposure. We, first, used MD simulations to compare the sulfur solvent accessibility of the cysteines in the wild-type form with those in two pathogenic mutants, L5S and V75D, 40 which overall did not show any significant change in the sulfur accessibility (Supporting Figure 10). We then constructed two different polyprotein constructs (Ig91−(γDc L5S ) 2 −Ig91 and Ig91−(γDc V75D ) 2 −Ig91) independently harboring each congenital cataract point mutation (Figure 4a). Pulling on the Ig91−(γDc L5S ) 2 −Ig91 polyprotein in an oxidizing (PBS) environment (Figure 4b) revealed that the most prevalent pathway (50%) corresponded to the unfolding trajectories featuring a reduction in the overall (γDc) 2 length, reminiscent of the formation of non-native disulfide bridges. The second most captured unfolding pathway (42%) followed the regular mechanical unfolding, and only 8% of the unfolding trajectories displayed the non-oxidative domain-swap dimerization unfolding pattern (Supporting Figure 11b). Noteworthy, for this L5S mutant, the mechanical stability of the Ntd is significantly decreased with respect to the wild-type protein, probably due to fact that the L5S mutation is located on the first β-strand of the Ntd (the protein's mechanical clamp), making both protein terminals mechanically indistinguishable (Supporting Figure 12). Pulling on the Ig91− (γDc V75D ) 2 −Ig91 protein (Figure 4c) gave rise to very similar results, whereby the predominant unfolding pathway (48%) Nano Letters pubs.acs.org/NanoLett Letter comprises unfolding trajectories featuring a shorter (γDc V75D ) 2 unfolding length, followed by the regular (36%) and domain swapped (16%) unfolding phenotypes (Supporting Figure  11c). Following a similar approach to that followed in Figure  3c, the measured ΔL c (γDc X ) 2 histogram reveals that, for both protein mutants, the proportion of intra-and intermolecular non-native disulfide formation is roughly the same (Supporting Figure 13). Combined, these experiments point toward an enticing scenario, where the lower thermodynamic stability of the Ntd domain of the protein mutants 38 would enhance a conformational change that would expose previously buried cysteines, increasing the probability of non-native disulfide bond formationa situation that becomes prevalent.

■ DISCUSSION
Whether beneficial or deleterious for the cell, 41,42 the detection of non-native, redox-regulated disulfide bridges has been particularly challenging. To date, most of the relevant studies use refined proteomics methods to tag 43 and capture these low-probability covalent bonds. Their highly dynamic nature, their fast reactivity, and their intimate relationship with the dynamically evolving protein structure necessitates the development of complementary experimental approaches.
Here, we use single-molecule force spectroscopy combined with protein engineering as a tool to capture non-native disulfide bonds by using molecular extensibility as their structural fingerprint. This methodology could be applied to virtually any protein, regardless of whether its physiological function is mechanical or not. As a proof-of-principle, we studied the redox-regulation of human γD-crystallin (γDc), the misfolding of which is directly related to eye's cataract. Our single-molecule experiments reveal the presence of both interand intramolecular non-native disulfide bonds in γDc. Pulling on the (Ig91−γDc) 2 polyprotein revealed the presence of nonnative intramolecular disulfide bonds, directly linked to the existence of partially folded conformations found in both γDc Ctd and γDc Ntd domains. The γDc Ctd has only two cysteines (cys 109 and cys 111 ), a priori limiting the possible non-native disulfide bonds to the one (cys 109 −cys 111 ) proposed by Serebryany et al. 44 However, the predominant intermediate conformation in γDc Ctd is characterized by ΔL c ∼9 nm, which would correspond to a "trapped length" much larger than that corresponding to the (cys 109 −cys 111 ) disulfide (Supporting Figure 14). To rationalize this, we conducted MD simulations, showing that the formation of the non-native disulfide bridge in the γDc Ctd can indeed induce a non-native partially folded protein conformation (Supporting Figure 15) compatible with the ΔL c ∼9 nm that we observe. This change in γDc Ctd conformation enforced by the formation of the non-native disulfide bridge is also likely accountable for the higher mechanical stability of the partially folded conformation (∼150 pN) when compared to the folded γDc Ctd structure (∼100 pN). A closer look into the γDc Ntd structure shows that the 4 cysteines are distributed evenly among the two Greek key motifs. Given that γDc Ntd shows a partially folded conformation characterized by a ΔL c ∼15 nm, a plausible scenario entails visiting a partially folded conformation whereby motif I is unfolded, while motif II remains folded (Supporting Figure  16). This partially folded conformation completely exposes three cysteines (cys 18 , cys 32 , and cys 41 ), which could explain the presence of both intramolecular and intermolecular disulfide bonds, the latter being uncovered when pulling on the Ig91−(γDc) 2 −Ig91 polyprotein. Given that our molecular dynamics simulations revealed that, in the folded form, only one cysteine in the Ctd is solvent-accessible, it is highly plausible that a conformational change (which we speculate involves the spontaneous unfolding of half of the Ntd) that exposes cryptic cysteines is a necessary step prior to disulfide bond formation. Interestingly, the partial unfolding of the Ntd motif I was suggested to swap with a neighboring γDc domain. 37 We, therefore, propose a plausible global scenario able to explain the three observed types of non-native conformations; if γDc remains in the native state, it exhibits a regular pattern of unfolding. However, once the protein undergoes a conformational change involving a "hinge" that opens Ntd motif I (Figure 5a), then two competing situations emerge. Either the protein swaps with a neighboring domain (eliciting the domain swapped conformations), or it forms non-native disulfide bonds (either intra-or intermolecular), marked by the "shorter" unfolding phenotype (Figure 5b).
An intrinsic limitation of our approach is that, given the number of cysteines present in each γDc monomer, and hence of possible disulfide cross-links, we do not have the resolution to identify which cysteine pairs are involved in each particular non-native disulfide bond formed between adjacent domains. The intrinsic design of the polyprotein chain is also likely to restrict the number of possible intermolecular disulfide bonds, if compared to the much larger number of available conformations when the interacting monomers are in solution. Conversely, the polyprotein approach has the advantage of offering unprecedented control over the onset of protein misfolding, occurring at the dimer level. Functionally, it remains unknown what is the relationship between non-native disulfides and domain swapped structures. In the presence of GSH ( Figure 3e) the population of "non-native disulfide trajectories" disappears, while the domain swapped population grows by essentially the same amount (from ∼20% to ∼50%), hence suggesting that these two conformations are in dynamic competition. As a first step toward relating these singlemolecule observations to bulk aggregation measurements, we measured the kinetics of light scattering of the Ig91−(γDc) 2 − Ig91 polyprotein in the absence and presence of reduced GSH (Figure 5c). Our results showed a drastic increase in the aggregation of the Ig91−(γDc) 2 −Ig91 polyprotein in the presence of GSH, strongly suggesting that the non-native disulfide bonds that we measured at the single-molecule level act as a safety mechanism that prevents protein aggregation, probably by limiting the misfolded, domain swapped conformation that leads to protein aggregation. From the physiological perspective, given that the eye lens are separate from the blood vessels and lack protein regeneration machinery, and that its glutathione concentration unavoidably diminishes over time, 45 it is tempting to speculate that the amount of non-native disulfide bonds, which is one of the key initial steps toward developing cataracts, 46 will increase over the life-span of the individual.
Altogether, our experiments demonstrate the power of single-molecule spectroscopy to capture non-native disulfide bonds, both intra-and also intermolecular, by using the protein extension as a conformational reporter. In the specific case of γDc, non-native disulfide formation is likely to follow from a stochastic yet well-defined conformational excursion of the Ntd to a partially folded conformation that exposes previously cryptic cysteines to the solvent, hence rendering them reactive. We anticipate that our combined experimental platform, encompassing protein engineering, single-molecule force spectroscopy experiments, MD simulations and classical biochemistry techniques can help uncover the dynamic nature of non-native protein disulfide bonds, of wide occurrence in nature.
Details of polyprotein engineering, single-molecule AFM setup and data analysis, light-scattering experiments, molecular dynamics simulations, and additional data sets (PDF) and S.G.M wrote the paper. All authors contributed to revising and editing the manuscript.